This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This research aims to determine the molecular mechanism of action of the A. nidulans transcriptional corepressor NmrA, a key component of an important regulatory system governing nutritional response. NmrA represses during nitrogen sufficiency the activity of the AreA GATA transcription factor. AreA activates expression of nitrogen metabolic genes during nitrogen limitation or nitrogen starvation. NmrA is thought to inhibit AreA activity by direct protein-protein interaction but the mechanism of NmrA action is not understood. This proposal will investigate how the corepressor NmrA regulates AreA activity. The key aims are (1) to establish whether NmrA acts as a regulatory NAD-dependent protein deacetylase enzyme, (2) to identify NmrA interactors by proteomics, and (3) to identify a new gene involved in NmrA-mediated repression. Objective 1: Does NmrA mediate repression by functioning as a regulatory enzyme? 1.1 Detection of NAD-dependent deacetylase activity using the Fluor-de-lys" substrate. Objective 2: Identification of proteins that interact with the NmrA repressor using a proteomics approach. 2.1 Biacore capture of proteins that interact with NmrA. Objective 3: Characterization of a suppressor of an NmrA overexpression phenotype. 3.1 Determination of the sequence change in a mutant affected in a novel gene affecting NmrA function by massively parallel sequencing. 3.2 Functional confirmation of mutations identified in the unknown gene. 3.3 Initial molecular genetic analysis of the new player in NmrA-mediated repression.